The long-term goal of our research is to establish well defined in vitro models for the regulation of cholesterol synthesis and to elucidate the molecular and biochemical mechanisms governing the level of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and cholesterol synthesis. The approach is to determine the site and mechanism through which cholesterol and oxygenated sterols regulate the level and activity of HMG-CoA reductase in established liver cell lines. The site and mechanism of cholesterol action will be established through immunochemical studies of HMG-CoA reductase synthesis, degradation and inactivation. The effects of cholesterol and oxygenated sterols on reductase synthesis, insertion into the microsomal membrane and degradation will be examined by immunochemical methods. The possibility that HMG-CoA reductase activity is modulated by ATP-Mg inactivation in vivo will be investigated using a technique we have recently developed for quanitate reactivation of ATP-Mg inactivated HMG-CoA reductase. The location, number and size of HMG-CoA reductase synthesizing polipomes will also be examined and the possible occurrence of a "signal" peptide on newly synthesized reductase will be examined. We shall carry out these investigations on both normal cells and on regulatory variants whose control of HMG-CoA reductase has been altered.